Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy.

In contrast to typical neuroleptic drugs, which have high affinities for dopamine D2 receptors, clozapine binds to multiple neurotransmitter receptors. The mechanisms responsible for its superior clinical efficacy over typical neuroleptics remain unknown. Using an automated genomics approach, total gene expression analysis (TOGA), we found an approximately threefold increase in the accumulation of the mRNA encoding apolipoprotein D (apoD) in mouse striatum in response to chronic treatment with clozapine. While in control animals, apoD is expressed predominantly in astrocytes, in situ hybridization and immunohistochemical studies indicated a substantial increase in apoD expression in neurons of the striatum, globus pallidus and thalamus after 2 weeks of clozapine treatment. Clozapine-induced increases in apoD expression were also observed in some white matter regions. These results suggest that apoD is a mediator in the mechanisms of clozapine and thus that deficiencies in aspects of lipid metabolism may be responsible for psychoses.

TOGA: an automated parsing technology for analyzing expression of nearly all genes.

We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases.

Refined structure of an intact IgG2a monoclonal antibody.

The structure of an intact, anti-canine lymphoma monoclonal antibody (Mab231) was determined by molecular replacement and refined in a triclinic cell to an R-value of 20.9%, using synchrotron diffraction data from 2.8 to 20 A resolution. All segments of the antibody, including the hinge region and carbohydrate component, are visible in electron density maps. There is no overall symmetry to the antibody, as the Fc is disposed in an entirely oblique manner with respect to the Fabs. The CH2 and CH3 domains do, however, possess a nearly exact, local 2-fold relationship. The Fab segments are related by a second, independent, local dyad axis, exact only with respect to constant domains. Variable domains exhibit no symmetry relationship as a consequence of the 16 degrees difference in Fab elbow angles. Variable domain pair associations VL:VH for the Fabs are virtually the same, and corresponding CDRs of the two Fabs also are nearly identical in structure. CDR-H3 displays the greatest difference. Hypervariable loops of both Fabs are involved in contacts with symmetry-related Fc segments at the CH2-CH3 switch junction, suggesting a "complex" structure. The hinge segment connecting Fabs with the Fc is quite extended and exhibits thermal factors indicative of a high degree of mobility. It consists of a well-defined upper hinge that partially maintains dyad symmetry and a fairly rigid core bounded above and below by fluid polypeptides that provide segmental flexibility. This structure represents the first visualization by X-ray analysis of a murine Fc segment, and its CH2 domains exhibit substantial rigid body conformational changes with respect to the human Fc used as an initial molecular replacement model. The oligosaccharides were found by difference Fourier syntheses to be very similar to those of the free human Fc fragment, although differences are present in the terminal residues. The detailed structure of the IgG presented here, and the distribution of effector binding sites, appears consistent with effector activation mechanisms involving translocation and/or aggregation of the Fc following antigen binding by the Fabs.

Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer.

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this study was to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a new fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1LA1 or the macrophage-tropic primary isolate HIV-1JR-FL. These cells fused with CD4+ target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1JR-FL gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1JR-FL and HIV-1LAI in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. Inhibition of HIV-1JR-FL envelope glycoprotein-mediated membrane fusion by soluble CD4 and CD4-IgG2 occurred at concentrations similar to those required to neutralize this virus. Interestingly, higher concentrations of these agents were required to inhibit HIV-1LAI envelope glycoprotein-mediated membrane fusion, in contrast to the greater sensitivity of HIV-1LAI virions to neutralization by soluble CD4 and CD4-IgG2. This finding suggests that the mechanisms of fusion inhibition and neutralization of HIV-1 are distinct.

Retinal degeneration slow (rds) in mouse results from simple insertion of a t haplotype-specific element into protein-coding exon II.

Retinal degeneration slow (rds) is a semidominant mutation of mice that causes dysplasia and degeneration of rod and cone photoreceptors. Mutations in RDS, the human ortholog of the rds gene, are responsible for several inherited retinal dystrophies including a subset of retinitis pigmentosa. The normal rds locus encodes rds/peripherin, an integral membrane glycoprotein present in outer segment discs. Genomic libraries from wildtype and rds/rds mice were screened with an rds cDNA, and phage lambda clones that span the normal and mutant loci were mapped. We show that in mice, rds is caused by the insertion into exon II of a 9.2-kb repetitive genomic element that is very similar to the t haplotype-specific element in the H-2 complex. The entire element is included in the RNA products of the mutant locus. We present evidence that rds in mice represents a null allele.

Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates.

CD4-IgG2 is a novel fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. This tetrameric protein is being developed as an immunoprophylactic agent to reduce the probability of infection following HIV-1 exposure, in settings such as occupational or perinatal exposure to the virus. CD4-IgG2 has been expressed in Chinese hamster ovary cells and is secreted as a fully assembled heterotetramer. The protein binds with nanomolar affinity to purified gp120 from both a laboratory-adapted strain and a primary isolate of HIV-1. Pharmacokinetic studies in rabbits demonstrated that CD4-IgG2 has a plasma terminal half-life greater than 1 day, compared with 15 min for soluble CD4 (sCD4). CD4-IgG2 does not bind to Fc receptors on the surface of U937 monocyte/macrophage cells. Compared to molecules that incorporate the Fc portion of IgG1, CD4-IgG2 has less potential to mediate functions such as antibody-dependent enhancement of infection or transplacental transmission of HIV-1. When tested in a virus-free HIV-1 envelope glycoprotein-mediated cell fusion assay, the tetrameric CD4-IgG2 molecule inhibited syncytium formation more effectively than monomeric sCD4 or a dimeric CD4-gamma 2 fusion protein. This suggests the protein will block cell-to-cell transmission of HIV-1. Moreover, CD4-IgG2 effectively neutralized a panel of laboratory-adapted strains and primary isolates of HIV-1, including strains with different tropisms and isolated from different stages of the disease, at concentrations that should be readily achieved in vivo.

Structure of an anti-idiotypic Fab against feline peritonitis virus-neutralizing antibody and a comparison with the complexed Fab.

The crystal structure of anti-idiotopic Fab 409.5.3, made against an E2 specific feline infectious peritonitis virus-neutralizing antibody 730.1.4, has been determined in its free from, at 2.9 A resolution by molecular replacement. This antibody, used as an immmunogen, elicits the production of anti-anti-idiotypic antibodies that in turn neutralize the virus. The structure of the uncomplexed Fab was refined using constrained-restrained least squares minimization and simulated annealing in combination with conjugate gradient techniques to a crystallographic R of 0.22 based on 16,482 unique reflections between 20.0 and 2.9 A. The free antiidiotypic Fab shows, when compared to its complexed form, a 5 degrees rotation of its variable light with respect to its variable heavy domain and rearrangement of complementarity determining region loops, which permits optimization of the stereocomplementarity between interacting molecules. This finding supports the induced fit hypothesis for antibody antigen interaction.

1G5: a calmodulin-binding, vesicle-associated, protein kinase-like protein enriched in forebrain neurites.

We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.

The alpha-helical rod domain of human lamins A and C contains a chromatin binding site.

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.

COS-7 cells stably transfected to express the human ETB receptor provide a useful screen for endothelin receptor antagonists.

Endothelin acts via specific membrane-bound receptors through signal transduction pathways that include increases in intracellular free calcium and inositol triphosphate generation. Two endothelin receptors have been cloned. The ETA receptor is ET-1 selective, and the ETB receptor is isopeptide nonselective. Both receptor subtypes are widely distributed throughout the body, although ETA receptors predominate in vascular smooth muscle, whereas ETB receptors predominate in the brain. The presence of mixed receptor subtypes makes functional screening of subtype-specific analogues difficult. A eukaryotic expression vector was constructed by inserting the cloned coding region of the human ETB receptor downstream from the Rous sarcoma promoter. COS-7 cells were transfected with this construct, and cell lines were isolated with stably integrated copies of the relevant gene. One line, 1C7, was shown to specifically bind 125I-ET-1. Scatchard analysis indicated a Kd value of 8.8 pM and a Bmax value of 1.02 pM/mg. ET-1 stimulated phosphoinositide hydrolysis in a dose-dependent manner, as did ET-3, sarafotoxin 6c, and [1,3,13,15Ala]ET-1, whereas BQ123, a selective ETA receptor antagonist, did not inhibit the action of ET-1. The transfected receptor stimulates phosphoinositide (PI) hydrolysis via a pertussis-sensitive pathway. Pretreatment of the membrane from 1C7 cells with dithio-bis-nitrobenzoic acid (DTNB) a negatively charged, nonpenetrating agent capable of oxidizing sulfhydryl groups, and N-ethyl-maleimide (NEM), a penetrating agent that causes irreversible alkylation of sulfhydryl groups, significantly reduces Bmax but has no effect on Kd. In whole cells, DTNB pretreatment abolishes the ability of ET-1 to stimulate PI hydrolysis.

The three-dimensional structure of an intact monoclonal antibody for canine lymphoma.

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.

An endoplasmic reticulum-specific cyclophilin.

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.

Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene.

Cyclic AMP is thought to act as an intracellular second messenger, mediating the physiological response of many cell types to extracellular signals. In the pituitary, growth hormone (GH)-producing cells (somatotrophs) proliferate and produce GH in response to hypothalamic GH-releasing factor, which binds a receptor that stimulates Gs protein activation of adenylyl cyclase. We have now determined whether somatotroph proliferation and GH production are stimulated by cAMP alone, or require concurrent, non-Gs-mediated induction of other regulatory molecules by designing a transgene to induce chronic supraphysiological concentrations of cAMP in somatotrophs. The rat GH promoter was used to express an intracellular form of cholera toxin, a non-cytotoxic and irreversible activator of Gs. Introduction of this transgene into mice caused gigantism, elevated serum GH levels, somatotroph proliferation and pituitary hyperplasia. These results support the direct triggering of these events by cAMP, and illustrate the utility of cholera toxin transgenes as a tool for physiological engineering.

Patterns of cerebral cortex mRNA expression.

A pool of 163 clones, isolated by screening 60,000 members of a Macaca fascicularis cerebral cortex cDNA library with a cortex-minus-cerebellum subtracted probe prepared by the phenol enhancement method, was analyzed by Northern blot hybridization studies. One hundred fifty-three of these clones corresponded to 22 RNAs whose abundance was at least 2-fold higher in cerebral cortex poly(A)+ RNA samples than in samples of cerebellar poly(A)+ RNA. Seven of these RNAs, represented by 131 clones, were undetectable in cerebellum. Only 10 of the 163 clones proved to be false positives. The abundance of several of these cortex-enriched RNAs was altered in Alzheimer's disease brains. Several RNAs that were present in cerebral cortex but undetectable in cerebellum were generally enriched in telencephalon, although none was restricted to the cortex. One of the cortex enriched RNAs, whose nucleotide sequence is presented, encoded monkey preprocholecystokinin. Overall, this study provides insights into the powers and limitations of subtractive hybridization and into the patterns of gene expression in the central nervous system.

Characterization of cloned measles virus mRNAs by in vitro transcription, translation, and immunoprecipitation.

A cDNA library was constructed from poly(A)+ RNA prepared from VERO cells infected with the Edmonston strain of measles virus. Clones corresponding to five major viral-specific transcripts were identified by northern blot hybridization analysis. Probes prepared from these five clones detected an additional five minor viral RNA transcripts. The sizes and hydridization patterns of these minor RNA species are consistent with their being bicistronic transcripts arising from a viral genomic template with the gene order 3'..NP-9/C-M-F-HA-(L)..5'. To assess the coding capacity of these cDNA clones they were inserted into pSP64, transcribed in vitro, the RNA was translated in reticulocyte lysates, and the protein was immunoprecipitated with specific antisera. From this analysis the genes for NP, P/C, M, F, and HA were identified. In vitro translation of natural mRNAs and SP6 transcripts of cDNAs consistently produced smaller polypeptides that appear to be initiated at internal AUGs. The relative abundance of these various cell-free translation products reflects the probability of translational initiation at the various in-frame AUGs. The patterns observed suggest that other factors besides sequences immediately flanking the AUGs have a significant effect on the selection of translational initiation sites. An increase in translational efficiency of the F transcript was achieved by removing 450 bases of the G-C-rich 5' noncoding region.

Cloning and in vitro expression of the measles virus matrix gene.

A cDNA library was prepared from Vero cells infected with the Edmonston strain of measles virus. A number of viral specific cDNA clones were isolated and characterized by Northern blot hybridization analysis. A cDNA clone containing a 1500 base pair insert which hybridizes to a viral specific transcript of approximately 1500 nucleotides was subcloned into pSP64 and used as an in vitro transcription template. The resulting RNA transcript was translated in a cell-free system, giving rise to a polypeptide which comigrates on polyacrylamide gels with the authentic measles virus matrix protein and is immunoprecipitated with antisera specific for the matrix protein.

Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system.

DNA coding for the alpha and beta subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the alpha subunit as a hybridization probe, we identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the alpha and beta subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to and released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase alpha and beta subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.